Potential of Y. lipolytica epoxide hydrolase for efficient production of enantiopure (R)-1,2-octanediol

Godase et al. AMB Express (2023) 13:77 https://doi.org/10.1186/s13568-023-01584-1 ORIGINAL ARTICLE AMB Express Open Access Potential of Y. lipolytica epoxide hydrolase for efficient production of enantiopure (R)-1,2octanediol Vijaya P. Godase1,2, V. Ravi Kumar3 and Ameeta Ravi Kumar1* Abstract The recombinant Yleh from a tropical marine yeast Yarrowia lipolytica NCIM 3589 exhibited a high epoxide hydrolase activity of 9.34 ± 1.80 µmol min-1 mg-1 protein towards 1,2-epoxyoctane (EO), at pH 8.0 and 30 °C. The reaction product was identified as 1,2-Octanediol (OD) by GC-MS using EO and H2O18 as substrate, affirming the functionality of Yleh as an epoxide hydrolase. For EO, the Km, Vmax, and kcat/Km values were 0.43 ± 0.017 mM, 0.042 ± 0.003 mM min-1, and 467.17 ± 39.43 mM-1 min-1, respectively. To optimize the reaction conditions for conversion of racemic EO by Yleh catalyst to enantiopure (R)-1,2-octanediol, initially, Response Surface Methodology was employed. Under optimized reaction conditions of 15 mM EO, 150 µg purified Yleh at 30 °C a maximal diol production of 7.11 mM was attained in a short span of 65 min with a yield of 47.4%. Green technology using deep eutectic solvents for the hydrophobic substrate (EO) were tested as co-solvents in Yleh catalyzed EO hydrolysis. Choline chloride-Glycerol, produced 9.08 mM OD with an increased OD yield of 60.5%. Thus, results showed that deep eutectic solvents could be a promising solvent for Yleh-catalyzed reactions making Yleh a potential biocatalyst for the biosynthesis of enantiopure synthons. *Correspondence: Ameeta Ravi Kumar ; Full list of author information is available at the end of the article © The Author(s) 2023. 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AMB Express (2023) 13:77 Page 2 of 15 Keywords Deep Eutectic solvents, Optimization, Recombinant epoxide hydrolase, Response surface methodology, Yarrowia lipolytica Introduction Vicinal diols and epoxides are important building blocks for the production of bioactive compounds used in cosmetics / personal care products and as raw materials for synthetic fiber, urethane compounds, water-based inks, insecticides, nanoparticles, etc. (Furuya et al. 2012; Effenberger et al. 2016; Sigg and Daniels 2020). Biological activity is highly stereoselective with the two enantiomers of the drug have different effects. Hence, regulatory authorities often warrant that chiral drugs be obtained optically pure to study pharmacological and toxicological properties. For example, EO and its vicinal diol, OD, are intermediates used in the asymmetric synthesis of highvalue pharmaceutical compounds. The diol is an essential building block in the production of steroids, β-blockers, adrenaline, nerve protectants, HIV protease inhibitors, leukotriene, insect pheromones, in treating head louse infestations and for the production of ferroelectric liquid crystals (Archelas and Furstoss 1998, 2001; Otto et al. 1988; Genzel et al. 2002; Burgess et al. 2012). Different methods have been used for the chemical synthesis of 1,2-octanediol using catalysts. Early processes for the preparation of vicinal diols and/or epoxides were carried out with unsaturated olefin compounds using alkali metals, alkaline earth metals, ammonium perrhenates, rhenium oxides as catalysts and hydrogen peroxide as an oxidation agent. Organic phosphoric acid esters and saturated ethers were used as solvents in pressure-controlled reactions occurring between 50 and 150 °C. The preparation of rhenium catalysts has its disadvantages with some of them being expensive and highly toxic. Additionally, yields of diols and epoxides were low with the formation of secondary oxidised products, such as ketones, carboxylic acids and/or polymeric condensation products (Warwel et al., 1994). Another common method to produce epoxy intermediates in the manufacture of 1,2-alkanediol can be carried out by combining 1-alkene with organic acid and hydrogen peroxide. Further, the epoxy compound is hydrolysed with alkali or an acid catalyst, followed by transesterification with alcohol to produce 1,2-alkanediol. However, the reaction is very slow and an alkyl organic acid ester may be generated as a by-product. Hence, higher concentrations of harsh chemicals, namely hydrogen peroxide, sulfuric acid, and benzene, are added to increase reactivity and lower by-product formation (Korean Patent KR102109133B1, 2020). A bifunctional Titanium silicalite (TS-1) catalyst system with both oxidative and Bronsted acid sites was designed by Prasetyoko (2006) for the consecutive transformation of 1-octene to octanediol with aqueous hydrogen peroxide as an oxidant. Another approach used active catalytic systems for alkene hydroxylation and cleavage by the highly toxic and expensive osmium tetroxide and transition metal catalysts such as ruthenium, iron, manganese, and cobalt to convert olefins into cis-1,2-diols. Palladium-catalyzed dihydroxylation and oxidative cleavage of olefins with oxygen as the sole oxidant and acid as an additive have also been studied (Wang and Jiang 2010). A more recent approach for obtaining diols from alkenes using electrolysis was studied using chemicals such as NaBr, THF, and Et4NBF4 with diol yields ranging from 10 to 56% but without enantioselectivity (Jud et al. 2021). Generally, these 1,2-diols are prepared by chemical processes having several limitations, as mentioned above, namely, the use of toxic chemicals, low substrate-to-catalyst ratios, and low efficiency with reactions carried out under extremes of temperature and pressure and waste generation. In this connection, mild, green, and clean processes such as the biocatalytic hydrolysis of racemic epoxides by epoxide hydrolases (EC 3.3.2.3) may prove advantageous. Amongst eukaryotic microbes, EHs from a few fungal/ yeast sources have been studied in detail, e.g., Aspergillus niger (Arand et al. 1999), Trichoderma reseei (Oliveira et al. 2016), Rhodotorula glutinis, Rhodosporidium toruloides and Saccharomyces cerevisiae (Smit 2004; Elfstrom and Widersten 2005). They exhibit different substrate specificities and enantio preferences, which are enzymedependent. (...truncated)